RESUMEN
The sensory cells that are responsible for hearing include the cochlear inner hair cells (IHCs) and outer hair cells (OHCs), with the OHCs being necessary for sound sensitivity and tuning1. Both cell types are thought to arise from common progenitors; however, our understanding of the factors that control the fate of IHCs and OHCs remains limited. Here we identify Ikzf2 (which encodes Helios) as an essential transcription factor in mice that is required for OHC functional maturation and hearing. Helios is expressed in postnatal mouse OHCs, and in the cello mouse model a point mutation in Ikzf2 causes early-onset sensorineural hearing loss. Ikzf2cello/cello OHCs have greatly reduced prestin-dependent electromotile activity, a hallmark of OHC functional maturation, and show reduced levels of crucial OHC-expressed genes such as Slc26a5 (which encodes prestin) and Ocm. Moreover, we show that ectopic expression of Ikzf2 in IHCs: induces the expression of OHC-specific genes; reduces the expression of canonical IHC genes; and confers electromotility to IHCs, demonstrating that Ikzf2 can partially shift the IHC transcriptome towards an OHC-like identity.
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Proteínas de Unión al ADN/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Células Ciliadas Auditivas Externas/citología , Células Ciliadas Auditivas Externas/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Transcriptoma/genética , Animales , Secuencia de Bases , Biomarcadores/metabolismo , Femenino , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Fukutin-related protein (FKRP, MIM ID 606596) variants cause a range of muscular dystrophies associated with hypo-glycosylation of the matrix receptor, α-dystroglycan. These disorders are almost exclusively caused by homozygous or compound heterozygous missense variants in the FKRP gene that encodes a ribitol phosphotransferase. To understand how seemingly diverse FKRP missense mutations may contribute to disease, we examined the synthesis, intracellular dynamics, and structural consequences of a panel of missense mutations that encompass the disease spectrum. Under non-reducing electrophoresis conditions, wild type FKRP appears to be monomeric whereas disease-causing FKRP mutants migrate as high molecular weight, disulfide-bonded aggregates. These results were recapitulated using cysteine-scanning mutagenesis suggesting that abnormal disulfide bonding may perturb FKRP folding. Using fluorescence recovery after photobleaching, we found that the intracellular mobility of most FKRP mutants in ATP-depleted cells is dramatically reduced but can, in most cases, be rescued with reducing agents. Mass spectrometry showed that wild type and mutant FKRP differentially associate with several endoplasmic reticulum (ER)-resident chaperones. Finally, structural modelling revealed that disease-associated FKRP missense variants affected the local environment of the protein in small but significant ways. These data demonstrate that protein misfolding contributes to the molecular pathophysiology of FKRP-deficient muscular dystrophies and suggest that molecules that rescue this folding defect could be used to treat these disorders.
RESUMEN
Hearing relies on mechanically gated ion channels present in the actin-rich stereocilia bundles at the apical surface of cochlear hair cells. Our knowledge of the mechanisms underlying the formation and maintenance of the sound-receptive structure is limited. Utilizing a large-scale forward genetic screen in mice, genome mapping and gene complementation tests, we identified Clrn2 as a new deafness gene. The Clrn2clarinet/clarinet mice (p.Trp4* mutation) exhibit a progressive, early-onset hearing loss, with no overt retinal deficits. Utilizing data from the UK Biobank study, we could show that CLRN2 is involved in human non-syndromic progressive hearing loss. Our in-depth morphological, molecular and functional investigations establish that while it is not required for initial formation of cochlear sensory hair cell stereocilia bundles, clarin-2 is critical for maintaining normal bundle integrity and functioning. In the differentiating hair bundles, lack of clarin-2 leads to loss of mechano-electrical transduction, followed by selective progressive loss of the transducing stereocilia. Together, our findings demonstrate a key role for clarin-2 in mammalian hearing, providing insights into the interplay between mechano-electrical transduction and stereocilia maintenance.
Asunto(s)
Pérdida Auditiva/metabolismo , Estereocilios/metabolismo , Adulto , Anciano , Animales , Estudios de Cohortes , Femenino , Células Ciliadas Auditivas/metabolismo , Audición , Pérdida Auditiva/genética , Pérdida Auditiva/fisiopatología , Humanos , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Persona de Mediana Edad , Estereocilios/genéticaRESUMEN
Kyphosis and scoliosis are common spinal disorders that occur as part of complex syndromes or as nonsyndromic, idiopathic diseases. Familial and twin studies implicate genetic involvement, although the causative genes for idiopathic kyphoscoliosis remain to be identified. To facilitate these studies, we investigated progeny of mice treated with the chemical mutagen N-ethyl-N-nitrosourea (ENU) and assessed them for morphological and radiographic abnormalities. This identified a mouse with kyphoscoliosis due to fused lumbar vertebrae, which was inherited as an autosomal dominant trait; the phenotype was designated as hereditary vertebral fusion (HVF) and the locus as Hvf. Micro-computed tomography (µCT) analysis confirmed the occurrence of nonsyndromic kyphoscoliosis due to fusion of lumbar vertebrae in HVF mice, consistent with a pattern of blocked vertebrae due to failure of segmentation. µCT scans also showed the lumbar vertebral column of HVF mice to have generalized disc narrowing, displacement with compression of the neural spine, and distorted transverse processes. Histology of lumbar vertebrae revealed HVF mice to have irregularly shaped vertebral bodies and displacement of intervertebral discs and ossification centers. Genetic mapping using a panel of single nucleotide polymorphic (SNP) loci arranged in chromosome sets and DNA samples from 23 HVF (eight males and 15 females) mice, localized Hvf to chromosome 4A3 and within a 5-megabase (Mb) region containing nine protein coding genes, two processed transcripts, three microRNAs, five small nuclear RNAs, three large intergenic noncoding RNAs, and 24 pseudogenes. However, genome sequence analysis in this interval did not identify any abnormalities in the coding exons, or exon-intron boundaries of any of these genes. Thus, our studies have established a mouse model for a monogenic form of nonsyndromic kyphoscoliosis due to fusion of lumbar vertebrae, and further identification of the underlying genetic defect will help elucidate the molecular mechanisms involved in kyphoscoliosis. © 2018 The Authors. JBMR Plus is published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.
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Insulin resistance in mice typically does not manifest as diabetes due to multiple compensatory mechanisms. Here, we present a novel digenic model of type 2 diabetes in mice heterozygous for a null allele of the insulin receptor and an N-ethyl-N-nitrosourea-induced alternative splice mutation in the regulatory protein phosphatase 2A (PP2A) subunit PPP2R2A. Inheritance of either allele independently results in insulin resistance but not overt diabetes. Doubly heterozygous mice exhibit progressive hyperglycemia, hyperinsulinemia, and impaired glucose tolerance from 12 weeks of age without significant increase in body weight. Alternative splicing of Ppp2r2a decreased PPP2R2A protein levels. This reduction in PPP2R2A containing PP2A phosphatase holoenzyme was associated with decreased serine/threonine protein kinase AKT protein levels. Ultimately, reduced insulin-stimulated phosphorylated AKT levels were observed, a result that was confirmed in Hepa1-6, C2C12, and differentiated 3T3-L1 cells knocked down using Ppp2r2a small interfering RNAs. Altered AKT signaling and expression of gluconeogenic genes in the fed state contributed to an insulin resistance and hyperglycemia phenotype. This model demonstrates how genetic changes with individually small phenotypic effects interact to cause diabetes and how differences in expression of hypomorphic alleles of PPP2R2A and potentially other regulatory proteins have deleterious effects and may therefore be relevant in determining diabetes risk.
Asunto(s)
Diabetes Mellitus Tipo 2/genética , Modelos Animales de Enfermedad , Haploinsuficiencia , Mutación , Proteína Fosfatasa 2/genética , Sitios de Empalme de ARN , Receptor de Insulina/genética , Alelos , Empalme Alternativo , Animales , Línea Celular , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/fisiopatología , Progresión de la Enfermedad , Heterocigoto , Resistencia a la Insulina , Masculino , Ratones , Ratones Mutantes , Proteína Fosfatasa 2/antagonistas & inhibidores , Proteína Fosfatasa 2/metabolismo , Interferencia de ARN , ARN Interferente Pequeño , Receptor de Insulina/metabolismo , Transducción de SeñalRESUMEN
Non-syndromic kyphosis is a common disorder that is associated with significant morbidity and has a strong genetic involvement; however, the causative genes remain to be identified, as such studies are hampered by genetic heterogeneity, small families and various modes of inheritance. To overcome these limitations, we investigated 12 week old progeny of mice treated with the chemical mutagen N-ethyl-N-nitrosourea (ENU) using phenotypic assessments including dysmorphology, radiography, and dual-energy X-ray absorptiometry. This identified a mouse with autosomal recessive kyphosis (KYLB). KYLB mice, when compared to unaffected littermates, had: thoraco-lumbar kyphosis, larger vertebrae, and increased body length and increased bone area. In addition, female KYLB mice had increases in bone mineral content and plasma alkaline phosphatase activity. Recombination mapping localized the Kylb locus to a 5.5Mb region on chromosome 15A1, which contained 51 genes, including the natriuretic peptide receptor 3 (Npr3) gene. DNA sequence analysis of Npr3 identified a missense mutation, Tyr209Asn, which introduced an N-linked glycosylation consensus sequence. Expression of wild-type NPR3 and the KYLB-associated Tyr209Asn NPR3 mutant in COS-7 cells demonstrated the mutant to be associated with abnormal N-linked glycosylation and retention in the endoplasmic reticulum that resulted in its absence from the plasma membrane. NPR3 is a decoy receptor for C-type natriuretic peptide (CNP), which also binds to NPR2 and stimulates mitogen-activated protein kinase (MAPK) signaling, thereby increasing the number and size of hypertrophic chondrocytes. Histomorphometric analysis of KYLB vertebrae and tibiae showed delayed endochondral ossification and expansion of the hypertrophic zones of the growth plates, and immunohistochemistry revealed increased p38 MAPK phosphorylation throughout the growth plates of KYLB vertebrae. Thus, we established a model of kyphosis due to a novel NPR3 mutation, in which loss of plasma membrane NPR3 expression results in increased MAPK pathway activation, causing elongation of the vertebrae and resulting in kyphosis.
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Cifosis/genética , Sistema de Señalización de MAP Quinasas , Mutación Missense , Receptores del Factor Natriurético Atrial/genética , Animales , Densidad Ósea , Células COS , Chlorocebus aethiops , Modelos Animales de Enfermedad , Etilnitrosourea/toxicidad , Femenino , Glicosilación , Cifosis/metabolismo , Cifosis/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Procesamiento Proteico-Postraduccional , Columna Vertebral/diagnóstico por imagen , Columna Vertebral/metabolismo , Tibia/diagnóstico por imagen , Tibia/metabolismoRESUMEN
Ectopic calcification (EC), which is the pathological deposition of calcium and phosphate in extra-skeletal tissues, may be associated with hypercalcaemic and hyperphosphataemic disorders, or it may occur in the absence of metabolic abnormalities. In addition, EC may be inherited as part of several monogenic disorders and studies of these have provided valuable insights into the metabolic pathways regulating mineral metabolism. For example, studies of tumoural calcinosis, a disorder characterised by hyperphosphataemia and progressive EC, have revealed mutations of fibroblast growth factor 23 (FGF23), polypeptide N-acetyl galactosaminyltransferase 3 (GALNT3) and klotho (KL), which are all part of a phosphate-regulating pathway. However, such studies in humans are limited by the lack of available large families with EC, and to facilitate such studies we assessed the progeny of mice treated with the chemical mutagen N-ethyl-N-nitrosourea (ENU) for EC. This identified two mutants with autosomal recessive forms of EC, and reduced lifespan, designated Ecalc1 and Ecalc2. Genetic mapping localized the Ecalc1 and Ecalc2 loci to a 11.0 Mb region on chromosome 5 that contained the klotho gene (Kl), and DNA sequence analysis identified nonsense (Gln203Stop) and missense (Ile604Asn) Kl mutations in Ecalc1 and Ecalc2 mice, respectively. The Gln203Stop mutation, located in KL1 domain, was severely hypomorphic and led to a 17-fold reduction of renal Kl expression. The Ile604Asn mutation, located in KL2 domain, was predicted to impair klotho protein stability and in vitro expression studies in COS-7 cells revealed endoplasmic reticulum retention of the Ile604Asn mutant. Further phenotype studies undertaken in Ecalc1 (kl203X/203X) mice demonstrated elevations in plasma concentrations of phosphate, FGF23 and 1,25-dihydroxyvitamin D. Thus, two allelic variants of Kl that develop EC and represent mouse models for tumoural calcinosis have been established.
Asunto(s)
Calcinosis/patología , Etilnitrosourea/toxicidad , Glucuronidasa/genética , Alelos , Secuencia de Aminoácidos , Animales , Células COS , Calcinosis/metabolismo , Chlorocebus aethiops , Codón sin Sentido , Modelos Animales de Enfermedad , Retículo Endoplásmico/metabolismo , Factor-23 de Crecimiento de Fibroblastos , Factores de Crecimiento de Fibroblastos/sangre , Factores de Crecimiento de Fibroblastos/genética , Sitios Genéticos , Genotipo , Glucuronidasa/química , Glucuronidasa/metabolismo , Humanos , Riñón/metabolismo , Proteínas Klotho , Longevidad/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Mutación Missense , N-Acetilgalactosaminiltransferasas/genética , Fenotipo , Fosfatos/sangre , Polimorfismo de Nucleótido Simple , Alineación de Secuencia , Vitamina D/análogos & derivados , Vitamina D/sangre , Polipéptido N-AcetilgalactosaminiltransferasaRESUMEN
Cushing's syndrome, which is characterized by excessive circulating glucocorticoid concentrations, may be due to ACTH-dependent or -independent causes that include anterior pituitary and adrenal cortical tumors, respectively. ACTH secretion is stimulated by CRH, and we report a mouse model for Cushing's syndrome due to an N-ethyl-N-nitrosourea (ENU) induced Crh mutation at -120 bp of the promoter region, which significantly increased luciferase reporter activity and was thus a gain-of-function mutation. Crh(-120/+) mice, when compared with wild-type littermates, had obesity, muscle wasting, thin skin, hair loss, and elevated plasma and urinary concentrations of corticosterone. In addition, Crh(-120/+) mice had hyperglycemia, hyperfructosaminemia, hyperinsulinemia, hypercholesterolemia, hypertriglyceridemia, and hyperleptinemia but normal adiponectin. Crh(-120/+) mice also had low bone mineral density, hypercalcemia, hypercalciuria, and decreased concentrations of plasma PTH and osteocalcin. Bone histomorphometry revealed Crh(-120/+) mice to have significant reductions in mineralizing surface area, mineral apposition, bone formation rates, osteoblast number, and the percentage of corticoendosteal bone covered by osteoblasts, which was accompanied by an increase in adipocytes in the bone marrow. Thus, a mouse model for Cushing's syndrome has been established, and this will help in further elucidating the pathophysiological effects of glucocorticoid excess and in evaluating treatments for corticosteroid-induced osteoporosis.
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Hormona Liberadora de Corticotropina/genética , Etilnitrosourea/química , Glucocorticoides/metabolismo , Mutación , Regiones Promotoras Genéticas , Animales , Composición Corporal , Huesos/metabolismo , Calcio/metabolismo , Línea Celular , Mapeo Cromosómico , Corticosterona/metabolismo , Síndrome de Cushing/genética , Modelos Animales de Enfermedad , Femenino , Metabolismo de los Lípidos , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Osteoblastos/metabolismo , Osteoporosis/metabolismoRESUMEN
Somatic and germline mutations in the dual zinc-finger transcription factor GATA3 are associated with breast cancers expressing the estrogen receptor (ER) and the autosomal dominant hypoparathyroidism-deafness-renal dysplasia syndrome, respectively. To elucidate the role of GATA3 in breast tumorigenesis, we investigated 40 breast cancers that expressed ER, for GATA3 mutations. Six different heterozygous GATA3 somatic mutations were identified in eight tumors, and these consisted of: a frameshifting deletion/insertion (944_945delGGinsAGC), an in-frame deletion of a key arginine residue (991_993delAGG), a seven-nucleotide frameshifting insertion (991_992insTGGAGGA), a frameshifting deletion (1196_1197delGA), and two frameshifting single nucleotide insertions (1224_1225insG found in three tumors and 1224_1225insA). Five of the eight mutations occurred in tumors that retained GATA3 immunostaining, indicating that absence of GATA3 immunostaining is an unreliable predictor of the presence of GATA3 mutations. Luciferase reporter assays, electrophoretic mobility shift assays, immunofluorescence, invasion and proliferation assays demonstrated that the GATA3 mutations resulted in loss (or reduction) of DNA binding, decrease in transactivational activity, and alterations in invasiveness but not proliferation. The 991_992insTGGAGGA (Arg330 frameshift) mutation led to a loss of nuclear localization, yet the 991_993delAGG (Arg330deletion) retained nuclear localization. Investigation of the putative nuclear localization signal (NLS) sites showed that the NLS of GATA3 does not conform to either a classical mono- or bi-partite signal, but contains multiple cooperative NLS elements residing around the N-terminal zinc-finger which comprises residues 264-288. Thus, approximately 20 % ER-positive breast cancers have somatic GATA3 mutations that lead to a loss of GATA3 transactivation activity and altered cell invasiveness.
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Neoplasias de la Mama/genética , Transformación Celular Neoplásica/genética , Factor de Transcripción GATA3/genética , Invasividad Neoplásica/genética , Secuencia de Aminoácidos , Neoplasias de la Mama/patología , Núcleo Celular/metabolismo , Núcleo Celular/patología , Femenino , Factor de Transcripción GATA3/metabolismo , Humanos , Células MCF-7 , Mutación , Invasividad Neoplásica/patología , Receptores de Estrógenos/genética , Activación Transcripcional/genéticaRESUMEN
The availability of high-throughput biochemical and imaging techniques that can be used on live mice has increased the possibility of undertaking longitudinal studies to characterize skeletal changes such as bone mineral content and density. Further characterization of bone morphology, bone quality, and bone strength can also be achieved by analyzing dissected bones using techniques that provide higher resolution. Thus, the combined use of high-throughput [e.g., biochemical analysis of plasma, radiography and dual-energy X-ray absorptiometry (DEXA)] and secondary phenotyping techniques (e.g., histology, histomorphometry, Faxitron digital X-ray point projection microradiography, biomechanical testing, and micro-computed tomography) can be utilized for comprehensive characterization of bone structure and quality and to elucidate the underlying molecular mechanisms giving rise to musculoskeletal disorders. Curr. Protoc. Mouse Biol. 2:365-400 © 2012 by John Wiley & Sons, Inc.
RESUMEN
Chronic kidney disease (CKD) is characterized by renal fibrosis that can lead to end-stage renal failure, and studies have supported a strong genetic influence on the risk of developing CKD. However, investigations of the underlying molecular mechanisms are hampered by the lack of suitable hereditary models in animals. We therefore sought to establish hereditary mouse models for CKD and renal fibrosis by investigating mice treated with the chemical mutagen N-ethyl-N-nitrosourea, and identified a mouse with autosomal recessive renal failure, designated RENF. Three-week old RENF mice were smaller than their littermates, whereas at birth they had been of similar size. RENF mice, at 4-weeks of age, had elevated concentrations of plasma urea and creatinine, indicating renal failure, which was associated with small and irregularly shaped kidneys. Genetic studies using DNA from 10 affected mice and 91 single nucleotide polymorphisms mapped the Renf locus to a 5.8 Mbp region on chromosome 17E1.3. DNA sequencing of the xanthine dehydrogenase (Xdh) gene revealed a nonsense mutation at codon 26 that co-segregated with affected RENF mice. The Xdh mutation resulted in loss of hepatic XDH and renal Cyclooxygenase-2 (COX-2) expression. XDH mutations in man cause xanthinuria with undetectable plasma uric acid levels and three RENF mice had plasma uric acid levels below the limit of detection. Histological analysis of RENF kidney sections revealed abnormal arrangement of glomeruli, intratubular casts, cellular infiltration in the interstitial space, and interstitial fibrosis. TUNEL analysis of RENF kidney sections showed extensive apoptosis predominantly affecting the tubules. Thus, we have established a mouse model for autosomal recessive early-onset renal failure due to a nonsense mutation in Xdh that is a model for xanthinuria in man. This mouse model could help to increase our understanding of the molecular mechanisms associated with renal fibrosis and the specific roles of XDH and uric acid.
Asunto(s)
Codón sin Sentido , Insuficiencia Renal/genética , Xantina Deshidrogenasa/genética , Animales , Análisis Químico de la Sangre , Mapeo Cromosómico , Cromosomas de los Mamíferos , Modelos Animales de Enfermedad , Femenino , Masculino , Ratones , Fenotipo , Insuficiencia Renal/metabolismo , Insuficiencia Renal/patologíaRESUMEN
Mutations of UDP-N-acetyl-alpha-D-galactosamine polypeptide N-acetyl galactosaminyl transferase 3 (GALNT3) result in familial tumoural calcinosis (FTC) and the hyperostosis-hyperphosphataemia syndrome (HHS), which are autosomal recessive disorders characterised by soft-tissue calcification and hyperphosphataemia. To facilitate in vivo studies of these heritable disorders of phosphate homeostasis, we embarked on establishing a mouse model by assessing progeny of mice treated with the chemical mutagen N-ethyl-N-nitrosourea (ENU), and identified a mutant mouse, TCAL, with autosomal recessive inheritance of ectopic calcification, which involved multiple tissues, and hyperphosphataemia; the phenotype was designated TCAL and the locus, Tcal. TCAL males were infertile with loss of Sertoli cells and spermatozoa, and increased testicular apoptosis. Genetic mapping localized Tcal to chromosome 2 (62.64-71.11 Mb) which contained the Galnt3. DNA sequence analysis identified a Galnt3 missense mutation (Trp589Arg) in TCAL mice. Transient transfection of wild-type and mutant Galnt3-enhanced green fluorescent protein (EGFP) constructs in COS-7 cells revealed endoplasmic reticulum retention of the Trp589Arg mutant and Western blot analysis of kidney homogenates demonstrated defective glycosylation of Galnt3 in Tcal/Tcal mice. Tcal/Tcal mice had normal plasma calcium and parathyroid hormone concentrations; decreased alkaline phosphatase activity and intact Fgf23 concentrations; and elevation of circulating 1,25-dihydroxyvitamin D. Quantitative reverse transcriptase-PCR (qRT-PCR) revealed that Tcal/Tcal mice had increased expression of Galnt3 and Fgf23 in bone, but that renal expression of Klotho, 25-hydroxyvitamin D-1α-hydroxylase (Cyp27b1), and the sodium-phosphate co-transporters type-IIa and -IIc was similar to that in wild-type mice. Thus, TCAL mice have the phenotypic features of FTC and HHS, and provide a model for these disorders of phosphate metabolism.
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Calcinosis/genética , Calcinosis/patología , Modelos Animales de Enfermedad , Hiperostosis Cortical Congénita/genética , Hiperostosis Cortical Congénita/patología , Hiperfosfatemia/genética , Hiperfosfatemia/patología , Mutación Missense/genética , N-Acetilgalactosaminiltransferasas/genética , Animales , Apoptosis/genética , Western Blotting , Huesos/metabolismo , Células COS , Chlorocebus aethiops , Etilnitrosourea/toxicidad , Factor-23 de Crecimiento de Fibroblastos , Factores de Crecimiento de Fibroblastos/metabolismo , Genes Recesivos/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Masculino , Ratones , Mutación Missense/efectos de los fármacos , N-Acetilgalactosaminiltransferasas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células de Sertoli/patología , Espermatozoides/patología , Testículo/citología , Polipéptido N-AcetilgalactosaminiltransferasaRESUMEN
Progeny of mice treated with the mutagen N-ethyl-N-nitrosourea (ENU) revealed a mouse, designated Longpockets (Lpk), with short humeri, abnormal vertebrae, and disorganized growth plates, features consistent with spondyloepiphyseal dysplasia congenita (SEDC). The Lpk phenotype was inherited as an autosomal dominant trait. Lpk/+ mice were viable and fertile and Lpk/Lpk mice died perinatally. Lpk was mapped to chromosome 15 and mutational analysis of likely candidates from the interval revealed a Col2a1 missense Ser1386Pro mutation. Transient transfection of wild-type and Ser1386Pro mutant Col2a1 c-Myc constructs in COS-7 cells and CH8 chondrocytes demonstrated abnormal processing and endoplasmic reticulum retention of the mutant protein. Histology revealed growth plate disorganization in 14-day-old Lpk/+ mice and embryonic cartilage from Lpk/+ and Lpk/Lpk mice had reduced safranin-O and type-II collagen staining in the extracellular matrix. The wild-type and Lpk/+ embryos had vertical columns of proliferating chondrocytes, whereas those in Lpk/Lpk mice were perpendicular to the direction of bone growth. Electron microscopy of cartilage from 18.5 dpc wild-type, Lpk/+, and Lpk/Lpk embryos revealed fewer and less elaborate collagen fibrils in the mutants, with enlarged vacuoles in the endoplasmic reticulum that contained amorphous inclusions. Micro-computed tomography (CT) scans of 12-week-old Lpk/+ mice revealed them to have decreased bone mineral density, and total bone volume, with erosions and osteophytes at the joints. Thus, an ENU mouse model with a Ser1386Pro mutation of the Col2a1 C-propeptide domain that results in abnormal collagen processing and phenotypic features consistent with SEDC and secondary osteoarthritis has been established.
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Colágeno Tipo II/genética , Mutación Missense/genética , Osteoartritis/complicaciones , Osteoartritis/genética , Osteocondrodisplasias/congénito , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Condrocitos/metabolismo , Condrocitos/patología , Condrocitos/ultraestructura , Cromosomas de los Mamíferos/genética , Colágeno Tipo II/química , Modelos Animales de Enfermedad , Embrión de Mamíferos/anomalías , Embrión de Mamíferos/patología , Sitios Genéticos/genética , Placa de Crecimiento/anomalías , Placa de Crecimiento/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Proteínas Mutantes/metabolismo , Tamaño de los Órganos , Osteocondrodisplasias/complicaciones , Osteocondrodisplasias/genética , Osteogénesis , Fenotipo , Mapeo Físico de Cromosoma , Procesamiento Proteico-PostraduccionalRESUMEN
Mutations in the fukutin-related protein gene (FKRP) are associated with a spectrum of diseases from mild limb-girdle muscular dystrophy type 2I to severe congenital muscular dystrophy type 1C, muscle-eye-brain disease (MEB), and Walker-Warburg syndrome (WWS). The effect of mutations on the transportation of the mutant proteins may constitute the underlying mechanisms for the pathogenesis of these diseases. Here we examined the subcellular localization of mouse and human normal and mutant FKRP proteins in cells and in muscle in vivo. Both normal human and mouse FKRPs localize in part of the Golgi apparatus in muscle fibers. Mutations in the FKRP gene invariably altered the localization of the protein, leading to endoplasmic reticulum retention within cells and diminished Golgi localization in muscle fibers. Our results therefore suggest that an individual missense point mutation can confer at least two independent effects on the protein, causing (1) reduction or loss of the presumed glycosyltransferase activity directly and (2) mislocalization that could further alter the function of the protein. The complexity of the effect of individual missense point mutations may partly explain the wide variation of the FKRP-related myopathies.
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Aparato de Golgi/metabolismo , Músculos/ultraestructura , Proteínas/metabolismo , Animales , Línea Celular Transformada , Cricetinae , Cricetulus , Expresión Génica/genética , Humanos , Proteínas Luminiscentes/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , Ratones Transgénicos , Músculos/metabolismo , Mutación/fisiología , Pentosiltransferasa , Transporte de Proteínas/genética , Proteínas/genética , Transfección/métodosRESUMEN
Myoclonus-dystonia syndrome (MDS) is a genetically heterogeneous disorder characterized by myoclonic jerks often seen in combination with dystonia and psychiatric co-morbidities and epilepsy. Mutations in the gene encoding epsilon-sarcoglycan (SGCE) have been found in some patients with MDS. SGCE is a maternally imprinted gene with the disease being inherited in an autosomal dominant pattern with reduced penetrance upon maternal transmission. In the central nervous system, epsilon-sarcoglycan is widely expressed in neurons of the cerebral cortex, basal ganglia, hippocampus, cerebellum and the olfactory bulb. epsilon-Sarcoglycan is located at the plasma membrane in neurons, muscle and transfected cells. To determine the effect of MDS-associated mutations on the function of epsilon-sarcoglycan we examined the biosynthesis and trafficking of wild-type and mutant proteins in cultured cells. In contrast to the wild-type protein, disease-associated epsilon-sarcoglycan missense mutations (H36P, H36R and L172R) produce proteins that are undetectable at the cell surface and are retained intracellularly. These mutant proteins become polyubiquitinated and are rapidly degraded by the proteasome. Furthermore, torsinA, that is mutated in DYT1 dystonia, a rare type of primary dystonia, binds to and promotes the degradation of epsilon-sarcoglycan mutants when both proteins are co-expressed. These data demonstrate that some MDS-associated mutations in SGCE impair trafficking of the mutant protein to the plasma membrane and suggest a role for torsinA and the ubiquitin proteasome system in the recognition and processing of misfolded epsilon-sarcoglycan.
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Trastornos Distónicos/genética , Chaperonas Moleculares/fisiología , Mutación Missense , Mioclonía/genética , Procesamiento Proteico-Postraduccional/fisiología , Sarcoglicanos/genética , Sarcoglicanos/metabolismo , Ubiquitina/metabolismo , Animales , Células COS , Membrana Celular/metabolismo , Células Cultivadas , Chlorocebus aethiops , Embrión de Mamíferos , Humanos , Ratones , Proteínas Mutantes/metabolismo , Transporte de Proteínas , Ratas , SíndromeRESUMEN
Mutations in the gene encoding fukutin-related protein (FKRP) cause a spectrum of diseases including congenital muscular dystrophy type 1C (MDC1C), limb girdle muscular dystrophy 2I (LGMD2I) and congenital muscular dystrophies (CMDs) with brain malformations and mental retardation. Although these diseases are associated with abnormal dystroglycan processing, the cellular consequences of the idiosyncratic FKRP mutations have not been determined. Here we show, in cultured cells, that FKRP mutants associated with the more severe disease phenotypes (S221R, A455D, P448L) are retained in the endoplasmic reticulum (ER), whereas the wild-type protein and the mutant L276I that causes LGMD2I are found predominantly in the Golgi apparatus. The ER-retained proteins have a shorter half-life than the wild-type FKRP and are preferentially degraded by the proteasome. Furthermore, calnexin binds preferentially to the ER-retained mutants suggesting that it may participate in the quality control pathway for FKRP. These data provide the first evidence that the ER-retention of mutant FKRP may play a role in the pathogenesis of CMD and potentially explain why the allelic disorder LGMD2I is milder, because the mutated protein is able to reach the Golgi apparatus.
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Retículo Endoplásmico/metabolismo , Distrofias Musculares/genética , Proteínas/genética , Animales , Células COS , Calnexina/metabolismo , Células Cultivadas , Chlorocebus aethiops , Humanos , Microscopía Confocal , Distrofias Musculares/metabolismo , Mutación , Pentosiltransferasa , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas/metabolismoRESUMEN
Recently, evidence has emerged that heptaspanning membrane or G protein-coupled receptors may be linked to intracellular proteins identified as regulators of receptor anchoring and signaling. Using a yeast two-hybrid screen, we identified alpha-actinin, a major F-actin-cross-linking protein, as a binding partner for the C-terminal domain of the adenosine A2A receptor (A2AR). Colocalization, co-immunoprecipitation, and pull-down experiments showed a close and specific interaction between A2AR and alpha-actinin in transfected HEK-293 cells and also in rat striatal tissue. A2AR activation by agonist induced the internalization of the receptor by a process that involved rapid beta-arrestin translocation from the cytoplasm to the cell surface. In the subsequent receptor traffic from the cell surface, the role of actin organization was shown to be crucial in transiently transfected HEK-293 cells, as actin depolymerization by cytochalasin D prevented its agonist-induced internalization. A2ADeltaCTR, a mutant version of A2AR that lacks the C-terminal domain and does not interact with alpha-actinin, was not able to internalize when activated by agonist. Interestingly, A2ADeltaCTR did not show aggregation or clustering after agonist stimulation, a process readily occurring with the wild-type receptor. These findings suggest an alpha-actinin-dependent association between the actin cytoskeleton and A2AR trafficking.